Immunoassays are a well established technique for detecting and quantifying analytes in samples. They are particularly useful for detecting and/or measuring substances in biological samples as an aid to disease diagnosis and prognosis, and for predicting a patient's response to therapy. Techniques such as the radioimmunoassay and enzyme immunoassay, which revolutionised diagnostic medicine, are based upon the detection of antibody-antigen interactions. Numerous detection systems are available, including the use of radio or enzyme labelled antigens, antibodies or complexes thereof. Many require incubation with specific substrates in order to measure the end-point calorimetrically, or by fluorescence.
Whilst these assays are sensitive, the detection systems are often complex, and therefore expensive. Typically, the assay systems require several washing steps, meaning that conventional assays are often unsuitable for point-of-care type assessment.
Agglutination immunoassays are well-known in the art, and rely upon agglutination of particles to which an antigen or antibody is bound to indicate the presence of the corresponding antibody or antigen in a sample. In one of the simpler forms of an agglutination assay, antibodies to a particular analyte are bound to a bead or other visible material (for example, polystyrene microparticles in the latex agglutination reaction). Typically, the antibody will be divalent, thus causing the latex beads to form clumps in the presence of an analyte. Such clumps indicate a positive result, and can be seen with the naked eye.
WO04/83859 describes a capillary based agglutination assay, comprising a capillary pathway which contains a reagent system capable of causing agglutination with the analyte. The reagent system comprises an antibody bound to either the capillary walls at a predetermined location, or antibody bound to beads which are placed in the capillary system at a predetermined location. Upon application to the capillary pathway, a sample flows along the pathway until it reaches the agglutination reagent system. If analyte is present, agglutination will occur, retarding further flow of the sample along the tube. Detection means for the presence of the sample at the downstream end of the pathway are effected after a predetermined time from application of the sample—if no sample can be detected, then analyte is present, indicating a positive result. This device uses latex beads as the agglutination means.
There is a growing need for assays to be performed closer to the patient, primarily to shorten the time taken to provide results. Such assays are known as Point-of-Care assays, and typically need to be robust and simple to perform since they are carried out in a non-laboratory setting, frequently by non-skilled staff. Ideally, they should be fully self-contained and require no ancillary equipment (with the possible exception of a reader). Point-of-Care assays need similar sensitivity to laboratory-based assays if they are to have any clinical use. However, conventional immunoassays often comprise complex protocols and detection systems, meaning that they are often unsuitable for point-of-care type use.
Specific Point-of-Care assays have been developed. The most common are lateral flow assays. Often, these are based on a labeled mobile component (e.g. coloured particle-labeled antibody), an immobilised component (e.g. antibody stripe or dot) and a membrane through which sample is caused to move by capillary action. In the presence of analyte, a “sandwich” is formed at the immobilised antibody capture zone, leading to development of a coloured line or dot. Conventional Lateral Flow Assays are exemplified by, for example, Unilever Patent Holdings B.V (U.S. Pat. No. 5,656,503). These assays specify an immobilised antibody capture zone, albeit in a lateral flow format as opposed to the radial format taught by Geigel et al (Clin Chem 28(9) pp 1894-8, 1982).
Lateral flow assays offer many advantages, including speed, convenience, and relatively low-cost. However, they have several drawbacks. The antibody is generally immobilised by adsorption onto the membrane, so variations in membrane and/or antibody batch can lead to variations in the amount of antibody immobilised. Further, some of the antibodies may be only loosely bound and can become mobile when the fluid front passes, leading to loss of signal. Also, since one antibody is immobilised, the only time for it to react with the analyte is as the sample flows past, so sensitivity can be reduced due to the short incubation time. It is also necessary to produce specific coated membranes for each analyte, thus increasing manufacturing costs.
Attempts have been made to address these disadvantages by avoiding the use of an immobilised capture antibody. For example, Miles (EP 297292), Hygeia Sciences (EP 310872), and Mizuho (EP 0962771) describe systems involving a membrane with a trapping zone in conjunction with 2 antibody-coated particles, one unlabeled but large such that it is trapped by the zone, the other small and labeled which can pass through the zone. In the presence of analyte, the small beads become bound to the trapped large beads leading to formation of a coloured line. Although these methods avoid the use of an immobilised capture antibody, they require two populations of antibody-coated particles in addition to a trapping zone. Frequently such particles are hydrophobic in nature, and thus can be caused to aggregate in a non-specific manner in the presence of biological fluids.
Others have attempted a simpler format, whereby antibody-coated particles capable of free movement through a membrane are caused to agglutinate in the presence of analyte such that their movement is halted. Such agglutination-based immunoassays are known in the art, and rely upon agglutination of particles to which an antigen or antibody is bound to indicate the presence of the corresponding antigen or antibody in a sample. In one of the simpler forms of an agglutination assay, antibodies to a particular analyte are bound to a bead or other visible material.
In particular, Amersham (U.S. Pat. No. 4,666,863) discloses a method for separating free and bound label by chromatographic means. In one variant, they teach separation of agglutinated and non-agglutinated antibody-coated coloured particles using flow along a membrane. Prior to separation, the reaction mixture is reacted with a cross-linking agent to stabilise the agglutinate. Daiichi (EP 293779) also discloses a coloured latex agglutination reaction, where agglutinated and non-agglutinated particles are separated by a capillary which allows non-agglutinated latex through but traps the aggregates. Kodak (EP 280559) describes an assay for multivalent analytes whereby in the absence of analyte label can pass through a filter, but in the presence of analyte an agglutinate is formed which is trapped. Akers (EP 556202) describes a system in which a test mixture is formed by contacting the sample with coloured particles having analyte-specific receptors on their surface. The test mixture is passed through a filter having pores which are larger than the coloured particles but smaller than the particle-analyte aggregates, thus causing trapping of the aggregates. Presence of aggregates from the mixture is determined by checking the colour of the filtrate. Genosis (U.S. Pat. No. 6,472,226) describes a lateral flow assay without immobilised antibody for very large analytes. They describe a 2-zone system, one having large pores and one having small pores, such that analyte can pass through the large pores but becomes trapped on reaching the zone of small pores. This is used in conjunction with a small label (e.g. gold sol) which can pass through both zones. In the presence of analyte, a fraction of the gold sol becomes bound to the analyte and becomes trapped at the small pore zone.
Common point of care assays are membrane lateral flow assays, generally used on urine samples. Urine contains a limited number of analytes, and so the application of these assays is restricted. To be used on whole blood, which contains a far greater range of analytes, filtration is usually necessary to remove the blood cells, as otherwise blockage and discolouration of the membrane would occur.
As a result, assay systems have been developed for use with whole blood, which do not require filtration of the blood. In U.S. Pat. No. 4,433,059, Chang discloses a non-capillary agglutination immunoassay in which two antibodies are covalently linked “tail to tail” to facilitate an autologous agglutination reaction utilizing particles endogenous to the sample. One antibody is specific for an antigen borne by an indicator substance, such as an erythrocyte. This antibody is univalent, and thus non-specific agglutination is avoided. The other antibody is divalent and specific for the analyte. In the presence of analyte, the conjugate antibody will cross-link the analyte and erythrocytes producing an agglutination of the erythrocytes.
Agen in various patent application (U.S. Pat. No. 5,413,913, U.S. Pat. No. 4,894,347, WO93/24630 and EP308242) describe a non-capillary agglutination system for use with whole blood, where the erythrocytes of the blood sample are used as the agglutination particles. The system requires the use of a conjugate comprising two antibodies or antibody fragments, one of which is directed against an erythrocyte antigen and the other of which is directed against a multi-epitopic analyte. In the presence of analyte, the antibody will agglutinate the erythrocytes.
Capillary based agglutination systems using whole blood as the sample have been previously disclosed, for example by U.S. Pat. No. 3,951,606 and WO 99/35497. These both give an indication of the presence/amount of analyte by determining either the location of the agglutinate or which capillary is blocked. Both of these assays are reliant upon the agglutination causing a total blocking of the capillary.
In the majority of current agglutination assays, the aggregation of particles is detected visually. However, a visual end-point is subjective and it is difficult to record the data electronically.
An important consideration is that, in the main, these agglutination-based assays are restricted to the detection of large analytes with multiple epitopes which enable the formation of large, stable agglutinates. Their effectiveness with smaller analytes having fewer epitopes, or where only a limited number of available epitopes are being used, can be compromised as the reduced number of binding events may result in a weakened aggregate and loss of sensitivity.
The present invention aims to address or ameliorate some or all of the above-mentioned problems associated with the assay systems of the prior art.